[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-10-25 to 2022-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997, corrected 26 June 2020

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

bis(diethylamino)methylidene-dibutylazanium;chloride

Cas Number:

89450-30-6

Molecular formula:

C17H38N3Cl

IUPAC Name:

bis(diethylamino)methylidene-dibutylazanium;chloride

Test material form:

solid

Details on test material:

Batch No.: EJ/V35/21

Method

Target gene:

Four strains of S. typhimurium and one strain of E. coli WP2 uvrA (pKM101) with the following characteristics were used:
TA98: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations
TA100: his G 46; rfa-; uvrB-; R-factor: base-pair substitutions
TA1535: his G 46; rfa-; uvrB-: base-pair substitutions
TA1537: his C 3076; rfa-; uvrB-: frame shift mutations
E. coli: WP2 uvrA (pKM101): trp-; uvrA-: base-pair substitutions
 
All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) genes (bacteria require biotin for growth).
The tester strains TA98, TA100 and E. coli contain the R-factor plasmid, pKM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pKM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms.
The tester strain E. coli WP2 uvrA (pKM101) carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

The bacteria most commonly used in these reverse mutation assays do not possess the enzyme system which, in mammals, is known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in the form of mammalian microsome enzyme activation mixture.
The S9 liver microsomal fraction was prepared at Eurofins Munich and obtained from Trinova Biochem GmbH, Gießen, Germany. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route (Eurofins Munich) and male Sprague Dawley rats were induced with phenobarbital/β-naphthoflavone (Trinova).
The following quality control determinations were performed:
• Eurofins Munich-prepared S9 Homogenate:
Quality control determinations performed by Eurofins Munich:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test
• Trinova Biochem GmbH-prepared S9 Homogenate:
Quality control determinations performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL (Eurofins Munich) and 5 mL (Trinova) and stored at ≤ -75 °C.

Test concentrations with justification for top dose:

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed at the following concentrations:
Experiment I:
3.16, 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(TA98, TA100)
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(TA1535, TA1537, E. coli WP2 uvrA (pKM101))
Experiment II:
0.100, 0.316, 1.0, 3.16, 10, 31.6, 100, 316 and 1000 µg/plate
(TA100 (without metabolic activation))
3.16, 10, 31.6, 100, 316, 1000 and 2500 µg/plate
(TA98, TA1535, TA1537 and E. coli WP2 uvrA (pKM101) (without metabolic activation))
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(All tester strains with metabolic activation)
As the results of the pre-experiment were in accordance with the criteria of validity (10.8), these were reported as a part of the main experiment I.

Vehicle / solvent:

The test item was dissolved in A. dest. and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

yes

Remarks:

A. dest

Negative solvent / vehicle controls:

yes

Remarks:

A. dest as in the negative control

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other:

Remarks:

dissolved in A. dest or DMSO

Details on test system and experimental conditions:

For the plate incorporation method, the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent or negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
For the pre-incubation method 100 µL of the test item-preparation is pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used (in a few cases two plates were evaluated).
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control [11].
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

above 1000 µg test concentration (Experiment II only)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

above 1000 µg test concentration without metabolic activation (based on Experiment II), above 2500 µg test concentration with metabolic activation (based on Experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

above 316 µg test concentration without metabolic activation (based on Experiment II), above 5000 µg test concentration with metabolic activation (based on Experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

above 1 µg test concentration without metabolic activation (based on Experiment II), above 1000 µg test concentration with metabolic activation (based on Experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

above 100 µg test concentration without metabolic activation (both Experiments), above 2500 µg test concentration with metabolic activation (based on Experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Test results are attached as pdf file

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Dibuthyltetraethylguanidinium chloride did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Dibuthyltetraethylguanidinium chloride is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item Dibuthyltetraethylguanidinium chloride was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I:

3.16, 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(TA98, TA100)

31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(TA1535, TA1537, E. coli WP2 uvrA (pKM101))

Experiment II:

0.100, 0.316, 1.0, 3.16, 10, 31.6, 100, 316 and 1000 µg/plate

(TA100 (without metabolic activation))

3.16, 10, 31.6, 100, 316, 1000 and 2500 µg/plate

(TA98, TA1535, TA1537 and E. coli WP2 uvrA (pKM101) (without metabolic activation))

31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(All tester strains with metabolic activation)

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

In experiment I toxic effects of the test item were observed at concentrations of 100 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation), depending on the particular tester strain.

In experiment II toxic effects of the test item were noted at concentrations of 1.0 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate (with metabolic activation), depending on the particular tester strain.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Dibuthyltetraethylguanidinium chloride at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met.